Background: Regulatory T cells (Treg) express high levels of lymphotoxin (LT) and bind the LT receptor (LTβR) on lymphatic endothelial cells (LEC). This receptor-ligand interaction is required for Treg afferent lymphatic trans-endothelial migration, and is important for islet allograft protection and survival. Activated CD4 T cells express TLR2, and its agonist Pam3CSK4 has been linked to T cell survival, proliferation, and differentiation, but hasn't been investigated to its effect on T cell migration. We hypothesized that TLR2 signaling may regulate migration through signaling interactions with TCR that augment LT expression and function.

Methods: Naive CD4 T cells were sorted from Foxp3GFP reporter mice and used to generate Treg and effector T cells (Teff). Treg and Teff were stimulated with TCR agonists or IL-2, with or without Pam3CSK4 (TLR2 agonist), LPS (TLR4), or R848 (TLR8). TCR or IL-2-mediated phosphatidyl inosine-3-kinase (PI3K)/Akt and MAPK signaling pathways in the stimulated cells were analyzed by immune blotting. T cells were analyzed for LT expression and for in vitro and in vivo lymphatic migration.

Results: Treg express much higher levels of LT than Teff. TCR and/or IL-2R signaling induce even more LT expression on Treg, but not on Teff. TLR2 signaling alone does not increase LT on Treg. However, TLR2 stimulation augments TCR and IL-2-induced LT expression on Treg, but not on Teff. Further, only TLR2, but not TLR4 or TLR8, activation enhances LT expression on Treg. TLR2 stimulation also promotes Treg, but not Teff, transendothelial migration across LEC toward CCL19. Phosphorylation of PI3K/Akt and ERK were induced in IL-2 stimulated Treg, and amplified by TLR2 co-stimulation. Blocking PI3K/Akt signaling abrogates the increase in LT expression and inhibits Treg migration. The peptide 2R9, which blocks the TLR2-MyD88/TIRAP signal axis, abolishes TLR2 stimulated LT expression on Treg and inhibits enhanced Treg lymphatic transmigration.

Conclusions: TCR and IL-2 stimulation both specifically increase Treg expression of LT. LT expression is further enhanced by TLR2 stimulation. Increased LT expression enhances Treg transmigration. TCR and IL-2 both signal via PI3K/Akt, and blocking PI3K prevents increased LT expression and enhanced migration. These results demonstrate a previously unexplored link between TCR/IL-2R and TLR2 signaling in Treg that leads to modulation of LT expression and regulation of T cell lymphatic migration. The TLR2 agonist and antagonist are potential tools for regulating Treg function in tissues and protection of the allograft.

CITATION INFORMATION: Piao W., Xiong Y., Li L., Saxena V., Bromberg J. TLR2 with TCR Signaling Mobilizes Treg Lymphatic Migration Am J Transplant. 2017;17 (suppl 3).

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